Messenger RNAs (mRNAs) are formed in the nuclei of eukaryotic cells by extensive post-transcriptional processing of primary RNA polymerase II transcripts. These transcripts are termed heterogeneous nuclear RNAs (hnRNAs) and they are associated with proteins throughout the time they are in the nucleus. The collective term for the proteins that bind hnRNAs, and which are not stable components of other classes of RNP complexes such as snRNPs, is hnRNP proteins. The hnRNP proteins are avid RNA-binding proteins and the processing of hnRNA in the nucleus takes place in hnRNA-protein (hnRNP) complexes. The significance of hnRNP proteins is that they influence the structure of hnRNAs and therefore their fate and processing into mRNAs. HnRNP proteins may also play crucial roles in the interaction of hnRNP complexes with other nuclear structures, in nucleo-cytoplasmic transport of mRNA, and they may have biochemical activity relevant to hnRNA metabolism. In addition, the hnRNP proteins are among the most abundant proteins in the nucleus in growing vertebrate cells, and hnRNP complexes are thus also of interest because they are major nuclear structures. Our goal is to understand in detail how the post-transcriptional portion of the pathway of gene expression operates in the cell. To do so we investigate the structure, function, and localization of the hnRNP and cytoplasmic mRNA-binding (mRNP) proteins and RNP complexes. Much progress has been made in these areas over the past several years but there is still a great deal of fundamental importance that remains to be learned. The focus of the research proposed in this grant application is the characterization of the hnRNP proteins, including their sequence, structure, RNA-binding activity, protein-protein interactions, assembly into complexes, biochemical activities, and function in mRNA biogenesis.